Seasonal influenza epidemics result in significant morbidity and mortality, and vaccine effectiveness is disappointingly low. The rapid evolution of influenza viruses is a major barrier to effective vaccines, as new strains with different antigenic properties are continuously generated within a remarkably large population of infected hosts. This problem was highlighted in the recent NIAID strategic plan for influenza and in PA-18-859, which highlight key knowledge gaps with respect to influenza virus evolution in individuals and its transmission between them. The long-term goal of this research is to elucidate the evolutionary and epidemiological dynamics of influenza viruses in naturally infected human hosts. The objectives of this project are to use state-of-the-art molecular approaches to define the host-level evolution of influenza and to identify correlates of infectivity and transmission. This project will combine intensive sampling of viruses, matched sera, and clinical data from naturally infected individuals and their contacts, which will allow for robust analyses of influenza evolution, transmission, and spread. The feasibility of this approach is supported by published preliminary data, which show that (i) serial sampling can be used to characterize the evolutionary dynamics of influenza viruses within naturally infected people; (ii) next generation sequencing (NGS) can be used to identify transmission pairs and to estimate the number of unique viral variants transmitted between individuals; (iii) temporal data on viral load and within- host genetic diversity can inform mathematical models of transmission. Detailed analyses of influenza evolution and transmission will be accomplished in three aims. (Aim 1) Identify the emergent antigenic variants that are positively selected with hosts. NGS of serially sampled within-host viral populations will be used to identify hemagglutinin and neuraminidase variants under positive selection within hosts and their relationship to host immune status. The impact of these mutations on viral antigenicity will be evaluated using sera from enrolled individuals. (Aim 2) Define the transmission bottleneck and the genetic variants that are transmitted. NGS of serial samples from index cases and household contacts will be used to define household transmission chains, the size of the transmission bottleneck, and which within host variants are transmitted. (Aim 3) Identify viral and host factors associated with host susceptibility, infectivity, and transmission. A strain-specific household transmission model, integrating molecular and epidemiologic data, will be used to estimate hazards of infection from the community and each infected household contact. This work is innovative, because it leverages the expertise of an existing productive team to combine state-of-the-art technologies for viral sequencing and sophisticated modeling approaches to study influenza virus evolution and transmission in natural infection. The proposed research is significant, because it will define the evolutionary dynamics of influenza viruses at the level of individual hosts in a community setting.